Many researchers have shown that over activation of the calcium-dependent proteases, the calpains, likely contributes to the cell death that occurs following brain injury. Therefore, it is critical to develop therapeutic agents to stem this over activation and reduce or prevent the subsequent cellular and tissue destruction. In preliminary work, we have used phage peptide display to identify a class of peptides that bind to calpain in a calcium-dependent manner. Interestingly, the representative peptide, LSEAL, has homology to conserved repetitive sequence found in calpastatin. Hence, we hypothesize that we have identified a novel class of calpastatin peptide mimetic. The goal of this proposal is to confirm and extend our initial observation that we have identified a novel family of calpain inhibitor for potential clinical use in the treatment of brain injury by carrying out the following specific aims: Specific Aim 1" Use phage peptide display to identify peptides that bind calpain in a calcium-dependent manner. We have already identified a candidate peptide, LSEAL. Additional libraries of increased stringency are to be used to find potentially more effective sequences. Specific Aim 2: To test the hypothesis that phage peptide display-derived peptides are novel and specific inhibitors of calpain activity with a mechanism similar to calpastatin. In preliminary work, we have found that a representative peptide from the initial library (LSEAL) is a potent inhibitor of calpain's actions on proteolysis of the microtubule-associated protein tau, a known calpain substrate. BIAcore technology will be used in addition to Elisa based assays to assay for calcium-dependent competitive binding between calpastatin, tau and LSEAL. We will also test additional calpain substrates. Specific Aim 3: To evaluate the hypothesis that LSEAL and related peptides act as novel calpain inhibitors in a cellular setting. In preliminary work, we have used cultured cortical neurons to demonstrate that cell death caused by increased cytosolic calcium mediated by either ionomycin or UV light is prevented by treatment with LSEAL and not by a scrambled sequence, negative control peptide, ELLAS. These studies will evaluate key aspects of LSEAL actions and provide important evidence to support the future development of this potentially new class of calpain inhibitor for use in the treatment of brain injury.